Reversibility of renal injury with cholesterol lowering in hyperlipidemic diabetic mice

Abstract

Hyperlipidemia is a risk factor for development and progression of diabetic nephropathy. However, it is not known if reduction of hyperlipidemia is protective against progression of disease. The goal of this study was to determine if reduction of hypercholesterolemia could limit progression of diabetic nephropathy. Diabetic and nondiabetic LDL receptor deficient (LDLR(-/-)) mice were fed diets containing either no cholesterol (0%) or high cholesterol (0.12%) for 36 weeks. One group each of diabetic and nondiabetic mice were fed the high-cholesterol diet for 26 weeks then changed to the 0% cholesterol diet for the last 10 weeks. Consumption of the high-cholesterol diet exacerbated the development of diabetic nephropathy with elevations in urine albumin excretion, glomerular and renal hypertrophy, and mesangial matrix expansion. Increased glomerular lipid and apolipoprotein B accumulation was found in diabetic mice that consumed the 0.12% cholesterol diet compared with other groups. However, diabetic mice that changed from the high-cholesterol diet to the 0% cholesterol diet for the last 10 weeks had lower urine albumin excretion and mesangial matrix expansion compared with mice that consumed the 0.12% cholesterol diet throughout. This suggests that hyperlipidemia causes continuous renal injury, and that lowering cholesterol levels by dietary means can improve renal function in diabetic LDLR(-/-) mice.

Fig. 1.

Effect of diabetes and diets on metabolic parameters. A: Blood glucose was measured from the tail vein in nonfasted mice at the indicated weeks of study using a glucometer. B: Mice were weighed at the indicated weeks of study. C: Plasma cholesterol was measured from nonfasted mice at the indicated weeks of study. Data shown is mean ± SEM for N = 7–14 per group. Gray symbols and lines indicate diabetic mice, black symbols and lines indicate control mice. Squares represent mice fed the 0% cholesterol diet, triangles represent mice fed the 0.12% cholesterol diet, and the dotted line represents mice that were changed from the 0.12% cholesterol diet to the 0% cholesterol diet at week 26. ^a represents P < 0.05 for effect of diabetes versus control. ^b represents P < 0.05 for effect of diets.

Fig. 2.

Effect of diabetes and diets on renal parameters. A: Urinary albumin excretion is expressed as mg albumin per g creatinine, and was measured from 24 h urine samples obtained from individual mice at the indicated weeks of study. Data shown is mean ± SEM for N = 7–14 per group. Gray symbols and lines indicate diabetic mice, black symbols and lines indicate control mice. Squares represent mice fed the 0% cholesterol diet, triangles represent mice fed the 0.12% cholesterol diet, and the dotted line represents mice that were changed from the 0.12% cholesterol diet to the 0% cholesterol diet at week 26. ^a represents P < 0.05 for effect of diabetes versus control. ^b represents P < 0.05 for effect of diets. B: Mesangial matrix was evaluated on renal sections stained with periodic acid Schiff (PAS). Shown are representative sections (from N = 7–14/group) from control or diabetic mice fed the 0% or 0.12% diets for 36 weeks, or the mice that changed diets at week 26. C: Mesangial matrix accumulation was scored using a semi-quantitative scale on at least 30 glomeruli/mouse sectioned through the glomerular tuft by two blinded observers (D.T. and L.R.T.) from N = 7–14/group). * represents P < 0.05 by Holm-Sidak pairwise comparison.

Fig. 3.

Effect of diabetes and diets on renal lipid accumulation. A: Frozen renal sections were stained with Oil Red O. Shown are representative sections from N = 7–14/group, magnified 400×. B: Paraffin sections were immunostained for apoB (red color product). Shown are representative sections from N = 7–14/group, magnified 400×. C: Total renal protein was analyzed by Western blot for apoB content. Each lane shows renal apoB from one mouse/group, representative of 6/group. Actin was used as the loading control. D: Western blots were analyzed by densitometry. Closed bars are control mice, open bars are diabetic mice. Shown is mean ± SEM from N = 6/group.